The primary objective of this investigation is to study the enzymatic mechanisms of DNA metabolism in Escherichia coli and bacteriophage - infected E. coli. Major emphasis will continue to be on the replication of phage T7 DNA both in vivo and in vitro. Bacteriophage induced protein, as well as host protein, involved in T7 DNA replication will be identified, purified and characterized in order to determine the molecular basis of their role in DNA replication. These proteins include the products of T7 genes 1.3, 2, 3, 4, 5 and 6, the T7 DNA binding protein, as well as E. coli thioredoxin and an E. coli protein involved in the initiation of replication. A major objective is the reconstitution, from individual components, of a replication system that minimizes DNA replication in vivo. A separate study involves modification-restriction in the T-even phage system. Studies on the in vitro restriction of DNA containing 5-hydroxymethyldeoxycytosine will be pursued by further purification of the r2, 4 protein of E. coli as well as other proteins and factors in this system. The purified enzymes will be used to determine the mechanism of restriction and to identify the restrictive sites in the DNA. Mutants of exonuclease VII will be used to establish the role of this enzyme in DNA metabolism in E. coli and in conjunction with biochemical studies to characterize further its physical and enzymatic properties.